Project protocol - Contents

• Workflow and sampling
• Equipment and supplies
• Reagents and solutions
• Procedure: Strain selection
• Procedure: Exercise programs
• Procedure: Indirect calorimetry
• Data

Workflow and sampling

Step	Procedure	Equipment	Data collected
1	Weigh mice before and after treatment	Scales	See McMullan6
2	Assess body composition before and after treatment	MRI	See McMullan6
3	Collect metabolic measurements and measure food and water intake before and after treatment	Indirect calorimetry	VO2, VCO2, RER, activity, food weight, water volume, heat production

Equipment and supplies

• Treadmill (Exer 3/6, Columbus Instruments, Columbus OH)
• Calorimetry system (PhenoMaster, TSE Systems, Chesterfield MO)

Reagents and solutions

• Disinfectant

Procedure: Strain selection

1. Four strains were selected based on the following criteria: 1) ability to run on treadmills, 2) fat response type to voluntary exercise, 3) endurance ability (MPD datasets: McMullan1, McMullan2, McMullan3, McMullan4; all data was collected in older females).
2. Selected strains were: CC002/Unc adverse fat responder, low endurance; CC027/GeniUnc adverse fat responder, high endurance; CC013/GeniUnc standard fat responder, low endurance; and CC037/TauUnc standard fat responder, high endurance.

Procedure: Exercise programs

1. High intensity interval training (HIIT): Five intervals with 80% max speed for four minutes, 20% max speed for one min, and ten 30 s transitions to decrease and increase speed between the different intensities.
2. Moderate intensity continuous training (MICT): Distance matched to the HIIT protocol and consists of ~43 min duration at 50% max speed.
3. No exercise (NE).
4. Mice complete 5 wks of exercise training on treadmills. Mice are acclimated to the treadmills for 3 days during the first week.
5. After acclimation, HIIT and MICT mice complete 4 wks of training, three times a week (all training occurs in the morning).
6. Compliance is tracked and mice with 50% or more non-compliant days are removed from statistical analysis. A mouse is considered non-compliant if it refuses or is not able to continue regular treadmill running despite extra stimulus from shock and/or prodding.

Procedure: Indirect calorimetry

1. Indirect calorimetry is assessed prior to any training and then again after the training period.
2. To acclimate to single housing, mice are individually housed for 24h prior to the start of the indirect calorimetry.
3. Mice are then individually housed in calorimetry cages for 24 hours and data are recorded every ~50 min for each calorimetry cage.
4. Food intake is calculated as the differential between baseline and post exercise food weights (g). Adjusted food intake is calculated as the food intake relative to the baseline body mass.
5. After, mice are returned to their assigned group housing.

Data collected by investigator

• Oxygen consumption
• Carbon dioxide output
• Respiratory Exchange Ratio (RER)
• Activity (beam breaks)
• Food weight
• Water volume
• Heat production